Genetic Factors Governing Susceptibilities to Severe Infections GSK-Chair of Infectious Diseases Pr Jean-Paul MIRA
Polymorphisms of Toll Like Receptors and Variability of Innate Immunity Institute of Cellular Pathology February 16, 2005 Pr. Jean-Paul MIRA Medical ICU & ICU & Dept Dept. of . of Cell Biology Cell Biology Medical Cochin University University Hospital Hospital & Cochin & Cochin Institute Institute, Paris, F , Paris, F Cochin
Septic Shock Epidemiology Septic Shock Epidemiology Annane D. Am J Respir Crit Care Med 2003;168:165–172
Variability of Severe Sepsis Phenotype
MIEUX COMPRENDRE MIEUX COMPRENDRE POUR MIEUX GUERIR POUR MIEUX GUERIR
« If it were not for the great variability among individuals medicine might as well be a science and not an art » Sir William Osler, 1892 1953 1953 2001- -2003 2003 2001 « Today we are learning the language in which God created life. Today we are learning the language in which God created life. « It will revolutionize the diagnosis, prevention and treatment of most, most, It will revolutionize the diagnosis, prevention and treatment of if not all human diseases. » » if not all human diseases. William J. Clinton, June 26, 2000
AS HUMANS, WE ALL SHARE AS HUMANS, WE ALL SHARE THE SAME BASIC GENES THE SAME BASIC GENES BUT… BUT…
Very small differences in genotype make beautiful differences in phenotype…
Small differences in genotype make big differences to phenotype
Evidences for a for a genetic genetic component to component to sepsis sepsis Evidences Animal Studies - Susceptibility/resistance to certain infection in mice
Mice Susceptibility to Infection to Infection with with Group A Group A Streptococci Streptococci Mice Susceptibility 10 3 cfu Strepto Subcutaneous d2 Goldman O. J Infect Dis 2003;187:854-61.
Evidences for a for a genetic genetic component to component to sepsis sepsis Evidences Animal Studies - Susceptibility/resistance to certain infection in mice Human Studies - Clinical Evidences - Ethnic Differences - Twin Studies - Adoptee Studies
Genetic and and environmental environmental influences influences Genetic on premature death premature death in in adult adoptees adult adoptees on Cause of Death Relative risk for the adoptee (Parent Dead before the age of 50) to die from the same cause All causes 1.71 Biologic 0.71 Adoptive Infection Biologic 5.8 Adoptive 0.73 Vascular 4.5 Biologic 3.1 Adoptive Sørensen TI, et al. NEJM 1988; 318:727-32.
STUDY NATURE AND OBJECTIVES STUDY NATURE AND OBJECTIVES STUDY METHODS Large, observational Population based associations (characterizing genetic risk and clinical usefulness) Genetic association with clinical outcomes Case control cohort comparisons Familial studies In vivo physiologic responses In vitro responsiveness Small, highly manipulated In vitro transfection (enhancing understanding of the biology of disease mechanisms) Knockout models
Genetic Polymorphisms and Severe Sepsis Gene Susceptibility and/or Outcome Mannose Binding Lectin Meningococcemia, Pneumococcemia Severe sepsis Toll-Like Receptor 4/2 Gram negative/positive Septic Shock Toll-Like Receptor 5 Legionnaire’s Disease CD14 Septic Shock FC γ RII Receptor Meningococcemia; Pneumococcemia TNF locus Meningococcemia;Septic Shock; Malaria IL-18 Severe Sepsis IL-10 Severe Sepsis, Meningococcemia IL-6 Severe sepsis Severe Sepsis IL-1 locus Viral Pneumonia IL-4 Septic shock Caspase12 PAI-1 Meningococcemia; Severe sepsis FactorV Leiden Meningococcemia; Severe sepsis
Pathogen Detection
Human Toll Toll- -like Receptors like Receptors Human • 10 10 Receptors Receptors (TLR1 (TLR1 – – TLR10) TLR10) • • Specific pathogen detection Specific pathogen detection • • Mediate the innate Mediate the innate immune immune response response • κ B - κ • Induce the inflammatory reaction Induce the inflammatory reaction (NF (NF- B) ) • • Induce Induce the the adaptative immune adaptative immune response response • 1 2 3 4 5 6 7 8 9 10 • Resolution Resolution of of the the inflammation ( inflammation (apoptosis apoptosis) ) 6 4 4 9 1 6 X X 3 3 • chromosome
Polymorphisms of the CD14 receptor
CD14 receptor and TLRs
CD14/159T and Susceptibility and Susceptibility to to Septic Shock Septic Shock CD14/159T CD14/159T increases sCD14 and mCD14 levels Control Septic Shock P<0.05 60 50 % Patients 40 30 20 10 -159CC -159CT -159TT Genotypes Le Van TD et al. J Immunol 2001;167:5838 Gibot S et al. Crit Care Med 2002; 30:969-73
CD14/159T and Mortality and Mortality to to Septic Shock Septic Shock CD14/159T Characteristics C/C C/T T/T p N=19 N=43 N=28 Age (mean ± SD) 53 ± 15 59 ± 13 59 ± 17 .18 SAPSII (mean ± SD) 53 ± 18 56 ± 21 60 ± 19 .21 OSF (mean ± SD) 3 ± 1.2 2.8 ± 0.9 3.1 ± 1 .42 Gibot S et al. Crit Care Med 2002; 30:969-73
CD14/159T and Mortality and Mortality to to Septic Shock Septic Shock CD14/159T Characteristics C/C C/T T/T p N=19 N=43 N=28 Age (mean ± SD) 53 ± 15 59 ± 13 59 ± 17 .18 SAPSII (mean ± SD) 53 ± 18 56 ± 21 60 ± 19 .21 OSF (mean ± SD) 3 ± 1.2 2.8 ± 0.9 3.1 ± 1 .42 Mortality (%) 26.3 58.1 71.4 <.0001 CD14/159TT RR= 5.1; 95%CI [3.2-7,9] Gibot S et al. Crit Care Med 2002; 30:969-73
Monocyte or LPS Dendritic cell Gram- Bacteria Flagellin Double-stranded ? RNA Lipoproteins CpG DNA Gram+ Bacteria Fungi TLR4 TLR3 TLR5 TLRx TLR9 TLR2 +/- TLR1/6 NF- κ B Signalisation Immunomodulatory genes Adaptative Response Tissue Injury Bacteria Lysis Septic Apoptosis Cellular Immunity Shock
- macrophages -/ /- in TLR4 - Impaired LPS LPS responsiveness responsiveness in TLR4 macrophages Impaired Hoshino et al., J. Immunol. 1999; 162:3749 Hoshino et al., J. Immunol. 1999; 162:3749
NF- -kB Activity kB Activity in in Transfected Transfected THP THP- -1 1 cells cells NF 100 NF κ B activity (L.U.x10 3 /mg) (-) LPS 80 (+) LPS 60 40 20 Wild type Thr399Ile Asp299Gly Arbour NC. Nature Genetics 2000;25:187
TLR4 mutation and and LPS LPS responsiveness responsiveness TLR4 mutation 40 IL-1 α (pg/ml) 20 WT/Asp299Gly WT/WT & Thr399Ile Arbour NC. Nature Genetics 2000;25:187
TLR4 Mutant Expression TLR4 Mutant Expression Human Airway Epithelia TLR4 WT TLR4 WT/299 Gly Arbour NC. Nature Genetics 2000;25:187
TLR4 Polymorphisms and Septic Shock Polymorphisms and Septic Shock TLR4 Gram negative Gram negative Control Control Septic shock Septic shock 30 patients mutated patients 25 20 % TLR4 mutated 15 10 % TLR4 5 Lorenz , Arch. Intern. Med. 2002 162:1028
TLR4 Variants and Predisposition and Predisposition to Gram to Gram Negative Sepsis Negative Sepsis TLR4 Variants With TLR4 TLR4 Without TLR4 TLR4 With Without infection negative infection Mutation Mutation Mutation Mutation 100 P = 0.004 80 % Gram negative 60 40 % Gram 20 Agnese DM, JID 2002; 186:1522
Monocyte or Lipopolysaccharide Dendritic cell Gram- Bacteria Flagellin Double-stranded RNA ? Lipoproteins CpG DNA Gram+ Bacteria Fungi TLR3 TLR4 TLR5 TLRx TLR9 TLR2 +/- TLR1/6 NF- κ B Signalisation Immunomodulatory genes Adaptative Response Tissue Injury Bacteria Lysis Septic Apoptosis Cellular Immunity Shock
-Knockout Mice Knockout Mice TLR2- TLR2
Echchannaoui H et al. JID 2002;186:798 TLR2 and Streptococcus pneumoniae meningitis TLR2 -/- WT
TLR2- -deficient mice and Gram positive infections deficient mice and Gram positive infections TLR2 IV challenge of lived Staphylococcus aureus Takeuchi et al., J. Immunol. 1999; 165:5392
TLR2- -deficient mice and Candida Infections deficient mice and Candida Infections TLR2 Candida albicans IP 50 x 10 6 Candida albicans IV 10 6 Villamon E., Microbes Infect 2004; 6:1-7
TLR2 Polymorphisms in Humans TLR2 Polymorphisms in Humans Tlr2 location: Chromosome 4q32 http://www.ncbi.nlm.nih.gov/entrez/query.fcqi http://innateimmunity.net 88 SNPs • 5’UTR: 26 SNPs • 3’UTR: 17 SNPs • Intron: 29 SNPs • Exon: 16 SNPs
Synonymous Non-synonymous TLR Structur Position rs number Position rs number 35 5743697 199 3804099 411(T/I) 5743699 213 5743698 e 556 (I/T) 5743702 450 3804100 579 (R/H) 5743703 541 5743700 542 5743701 631 (P/H) 5743704 677 (R/W) 5743706 707 IIPGA-TLR2-31410 715 (Y/N) 5743707 715 (Y/Amb) 753 (R/Q) 5743708 781 5743709
Location of R753 in the TIR domain
R753Q Polymorphism in the TLR2 gene R753Q Polymorphism in the TLR2 gene 743 743 765 765 Human TLR2 K A I P Q R P C K L R R K I M N T K T Y L E W P K I M N T K T Y L E W P Human TLR2 K A I P Q R P C K L Mouse TLR2 K A I P Q R P C K L R R K I M N T K T Y L E W P K I M N T K T Y L E W P Mouse TLR2 K A I P Q R P C K L kB activation (x fold increase) -kB activation (x fold increase) Q Q No stimulation No stimulation 6 6 Lipoproteins Lipoproteins 5 5 4 4 3 3 2 2 1 1 NF- NF TLR2 wt TLR2 R753Q TLR2 wt TLR2 R753Q Lorenz, Infect. Immun. 2OOO Lorenz, Infect. Immun. 2OOO
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